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| United States Patent |
5,096,922
|
|
Reichenbach
, et al.
|
March 17, 1992
|
Macrocyclic antibodies
Abstract
The invention relates to cyclic antibiotics of formula
##STR1##
wherein the prefix R represents that the substituents on the adjacent
carbon atom are in the R configuration; the prefix S represents that the
substituents on the adjacent carbon atom are in the S configuration;
R.sub.1 is hydrogen or hydroxy and R.sub.2 is hydroxy and salts thereof,
especially pharmaceutically acceptable salts.
The compounds of formula I are prepared by fermentation using the strain So
ce 12 (NCIB 12134) of the species Sorangium cellulosum and are effective
antibiotics.
| Inventors:
|
Reichenbach; Hans (Wolfenbuttel, DE);
Hofle; Gerhard (Brunswick, DE);
Augustiniak; Hermann (Wolfenbuttel, DE);
Bedorf; Norbert (Konigslutter, DE);
Gerth; Klaus (Brunswick, DE);
Irschik; Herbert (Wolfenbuttel, DE);
Jansen; Rolf (Brunswick, DE);
Kunze; Brigitte (Brunswick, DE);
Schomburg; Dietmar (Brunswick, DE);
Steinmetz; Heinrich (Hildesheim-Sorsum, DE);
Trowitzsch-Kienast; Wolfram (Brunswick, DE);
Wray; Victor (Brunswick, DE)
|
| Assignee:
|
Gesellschaft fur Biotechnologische Forschung mbH (Brunswick, DE)
|
| Appl. No.:
|
512963 |
| Filed:
|
April 23, 1990 |
Foreign Application Priority Data
| Current U.S. Class: |
514/450; 536/7.1; 549/267 |
| Intern'l Class: |
A61K 031/335; C07D 321/00 |
| Field of Search: |
536/6.5,7.1
549/264,265,267,268
514/453,450
435/76,822
|
References Cited
| Foreign Patent Documents |
| 0272667 | Jun., 1988 | EP | 536/6.
|
Primary Examiner: Brown; Johnnie R.
Assistant Examiner: Peselev; Elli
Attorney, Agent or Firm: Kane, Dalsimer, Sullivan, Kurucz, Levy, Eisele and Richard
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a division of our U.S. patent application Ser. No.
907,446 filed Sept. 11, 1986 and now issued as U.S. Pat. No. 4,987,072.
Claims
What is claimed is:
1. A compound of the formula
##STR3##
wherein the prefix R represents that the substituents on the adjacent
carbon atom are in the R configuration; the prefix S represents that the
substituents on the adjacent carbon atom are in the S configuration;
R.sub.1 is hydrogen or hydroxy and R.sub.2 is hydroxy and, if R.sub.1 is
hydroxy, the C-22 atom has the S-configuration, or a pharmaceutically
acceptable salt thereof.
2. A compound of formula I
##STR4##
wherein R.sub.1 is hydroxy and R.sub.2 is hydroxy (sorangicin A) and the
C-22 atom has the S-configuration.
3. The ethyl acetate solvate of a compound as claimed in claim 1.
4. A compound of formula I, wherein R.sub.1 is hydrogen, R.sub.2 is
hydroxy.
5. A pharmaceutical composition, which comprises an inorganic or organic,
solid or liquid, pharmaceutically acceptable excipient and an effective
amount for the treatment of susceptible infections of a compound of
formula I as defined in claim 1.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to novel macrocyclic compounds, in particular
to sorangicin A, to a fermentation process for the preparation of said
compounds using a novel microorganism of the species Soranguim cellulosum
(also known as Polyangium cellulosum) and to the novel microorganism
itself. The invention further relates to pharmaceutical compositions which
contain the novel compounds, to a therapeutic method comprising the use of
said compounds as antibiotics, and to the use thereof for the preparation
of pharmaceutical compositions.
SUMMARY OF THE INVENTION
Specifically, the present invention relates to macrocyclic compounds of
formula
##STR2##
wherein the prefix R represents that the substituents on the adjacent
carbon atom are in the R configuration; the prefix S represents that the
substituents on the adjacent carbon atom are in the S configuration;
R.sub.1 is hydrogen or hydroxy and R.sub.2 is hydroxy or
.beta.-glucopyranosyloxy, and to solvates and salts thereof, in particular
to pharmaceutically acceptable acid addition salts thereof.
The configuration of the substituents at the chiral C-atoms has been
ascertained by means of X-ray structural analysis of the crystalline
compound of formula I, sorangicin A (R.sub.1 =OH, R.sub.2 =OH), and
determined by the sequence rules of Cahn, Ingold and Prelog. If R.sub.1 is
hydroxy, the C-22 atom has the S-configuration.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates in particular to the main component of formula I
obtainable by fermentation and designated as sorangicin A (R.sub.1 =OH,
R.sub.2 =OH, C-22:S), and to the secondary components comprising
sorangicin B (R.sub.1 =H, R.sub.2 =OH), sorangioside A (R.sub.1 =OH),
R.sub.2 =.beta.-glucopyranosyloxy, C-22:S), and sorangioside B (R.sub.1
=H, R.sub.2 =.beta.-glucopyranosyloxy).
A further preferred object of the invention is the deposited microorganism
of the species Sorangium cellulosum (Polyanguim cellulosum), which is
described hereinafter and is employed for the preparation of the main
component, sorangicin A, by fermentation, as well as the of the secondary
components cited above. The taxa "Sorangium cellulosum" as used herein is
synonymous with "Polyangium cellulosum", which terms are used
interchangeably in the scientific community.
The compounds of formula I may be in the form of solvates. For example,
sorangicin A can be isolated in the form of the crystalline ethyl acetate
solvate.
Salts are preferably the pharmaceutically acceptable or non-toxic salts of
compounds of formula I. Such salts are in particular suitable alkali metal
salts such as sodium or potassium salts, or alkaline earth metal salts,
e.g. magnesium or calcium salts, and also zinc salts or ammonium salts,
including those salts that are formed with organic amines such as
unsubstituted or hydroxy-substituted mono-, di- or trialkylamines, e.g.
diethylamine, bis(2-hydroxyethyl)amine, triethylamine,
N,N-dimethyl-N-(2-hydroxyethyl)amine, tris(2-hydroxyethyl)amine or
N-methyl-D-glucamine. Pharmaceutically unacceptable salts, e.g. sparingly
soluble and/or readily crystallising salts can be used for isolation and
purification.
The compounds of formula I, especially the main component, sorangicin A,
which is obtainable by fermentation, as well as pharmaceutically
acceptable salts thereof, are effective antibiotics for use in human and
veterinary medicine. Their activity is directed against coccae, bacteria
and viruses. Controllable viruses are in particular those requiring
reverse transcriptase for replication (retroviruses).
For example, MIC (minimum innhibitory concentration) values have been found
in vitro in the agar dilution test (H. M. Ericsson and S. C. Sherris, Acta
Path. Microb. Scand. Section B, Suppl. No. 217, 1-90, 1971) which are in
the range from 0.01 to 2 .mu.g/ml for aerobic, gram-positive and
gram-negative coccae, e.g. Staphylococcus aureus, Neisseria gonorrhoeae,
N. meningitidis, Streptococcus spp., and from c. 0.01 to 16 .mu.g/ml for
Haemophilus influenzae, Pseudomonas aeruginosae, enterobacteria such as
Escherichia coli, Klebsiella pneumoniae, Proteus spp., Enterobacter
cloacae, Serratia marcescens or anaerobes, e.g. Bacteroides fragilis or
Clostridium perfingen. MIC values in the range from 0.5 to 8 .mu.g/ml have
been found for mycobacteria, e.g. M. tuberculosis and atypical
mycobacteria.
In the in vitro test on human polymorphonuclear leucocytes and
Staphylococcus aureus Strain Wood 46, sorangicin A is effective against
bacteria that occur intracellularly, e.g. staphylococcae. The substance
has a bactericidal activity within leucocytes.
Proof of the antiviral activity of the compounds of formula I against
retroviruses is shown by in vitro and in vivo tests, but especially by an
in vitro test system in which the inhibition of the reverse transcriptase
enzyme is determined quantitatively by said compounds.
On poly A-oligo (dT), radioactively labelled TPP is hybridised with reverse
transcriptase. The latent radioactivity is measured and serves as
reference for the activity of the reverse transcriptase.
The ID.sub.50 of the compounds of formula I is determined by using them in
the test system in different concentrations.
Sorangicin A is active against reverse transcriptase of Moloney Murine
leucaemia virus; the ID.sub.50 [dose that inhibits the RT activity by 50%;
method of Wu et al., Proc. Natl. Acad. Sci., 69, 3820 (1972)] for the
reverse transcriptase of MLV is c. 7 .mu.g/ml.
Compounds of formula I are prepared by culturing the strain So ce 12 (NCIB
12134) of the species Sorangium cellulosum, or a mutant derived from said
strain that produces compounds of formula I, in a culture containing a
source of carbon and nitrogen and essential inorganic salts, in the
temperature range from about 15.degree. to 40.degree. C. and at a pH in
the range from about 4.0 to 8.0, under aerobic conditions and, if desired,
isolating the resultant compounds of formula I and/or converting a
resultant compound into a salt and/or a resultant salt into the free
compound or into another salt.
The microorganism So ce 12 of the species Sorangium cellulosum was obtained
from a soil sample from the region of Xcaret, Yucatan Peninsula, Mexico.
It can be classified as a strain of the myxobacterium, Sorangium
cellulosum.
The strain So ce 12 was deposited on 1st Aug. 1985 with The National
Collections of Industrial and Marine Bacteria Ltd. in Aberdeen, Scotland,
UK, under the number NCIB 12134, in accordance with the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. A viability
certificate was issued by this depository authority on 12th Aug. 1985. The
public has access to the deposit.
ISOLATION AND DESCRIPTION OF SO CE 12
The soil sample was placed on filter paper over Stan 21 agar (0.1% of
K.sub.2 HPO.sub.4, 0.1% of KNO.sub.3, 0.1% of MgSO.sub.4.7H.sub.2 O, 0.01%
of MnSO.sub.4.7H.sub.2 O, 0.1% of CaCl.sub.2.2H.sub.2 O, 0.02% FeCl.sub.3,
standard trace element solution (consisting e.g. of 20 mg/l each of
Na.sub.2 MoO.sub.4.2H.sub.2 O, Na.sub.2 B.sub.4 O.sub.7. 10H.sub.2 O,
MnSO.sub.4. H.sub.2 O and CuSO.sub.4.H.sub.2 O), 0.002% of yeast extract
(Difco), 1% of agar, 25 mg/l of cycloheximide (actidione), and incubated
for 3 weeks at 30.degree. C.
Swarm cell colonies with fruiting bodies were again inoculated on filter
paper over Stan 21 agar. Amoebae were removed by aeration with a 5%
aqueous solution of ammonia. The fruiting bodies in EBS solution (0.5% of
peptone from casein, tryptic, ex Merck Darmstadt, Germany, 0.5% of
proteosepepton, Difco, 0.1% of peptone from meat extract, Merck, 0.1% of
yeast extract, Difco, sterilised in an autoclave) were then kept overnight
at 4.degree. C. with AB-1 antibiotic solution (20 mg of chloroamphenicol,
30 mg of steptomycin sulfate, 25 mg of tetracycline hydrochloride, 20 mg
of cephalotin, dissolved in 50 ml of water and sterilised by filtration)
and subsequently streaked out on vy/2 agar (0.5% of baker's yeast, based
on fresh weight, 0.1% of CaCl.sub.2.2H.sub.2 O, 1.5% of agar, pH 7.2).
Developing, pure swarm cell colonies were further cultivated on the same
medium and examined on filter paper over Stan 21 agar for identity with
the starting isolate.
The vegetative cells are cylindrical rods with round ends, mostly c. 1
.mu.m wide and 3-6 .mu.m long. Under a phase contrast microscope they
appear dark. They move by gliding. On many nutrient media, the organism
forms a multitude of fruiting bodies, e.g. on filter paper over mineral
salt agar (Stan 21 agar). The fruiting bodies resemble rust-coloured pads
and consist of a substantially large number of sporangioles, spherical or,
through mutual flattening, polyhedral structures with a solid wall 20 to
30 .mu.m in diameter.
The sporangioles contain myxospores, rod-shaped permanent cells of similar
shape and size as the vegetative cells, but readily refractive.
The strain So ce 12 can spontaneously form mutants (natural mutants) or
artificial mutants can be prepared which, like the natural strain, produce
antibiotic compounds of formula I.
Such mutants can be produced by chemical means, e.g. by treatment with
certain guanidine derivatives, e.g. N-methyl-N'-nitro-N-nitrosoguanidine,
or with an alkali metal nitrite such as sodium nitrite, or by physical
means, e.g. with energy-rich radiation such as ultraviolet, X-ray or
radioactive radiation.
The culture eligible for culturing must contain a source of carbon and of
nitrogen as well as essential inorganic salts. Examples of suitable carbon
sources are: assimilable carbohydrates, e.g. D-glucose, D-xylose,
L-arabinose, D-frutose, maltose, maltotriose, and starch. Suitable
nitrogen sources are: amino acids, peptides and proteins as well as their
degradation products such as peptone or tryptone, and also meat extracts,
cereal flour, e.g. corn or wheat, beans, especially soybeans, seeds, e.g.
of cotton plants, distillation residues from alcohol production, yeast
extracts and the like, and ammonium salts and nitrates. As essential
inorganic salts the nutrient solution may contain e.g. chlorides,
carbonates, sulfates, phosphates of alkali metals or alkaline earth
metals, e.g. sodium, potassium, magnesium, calcium, iron, zinc, manganese,
molybdenum and copper. Culturing is preferably carried out in liquid
culture media, most preferably in aqueous culture media.
A particularly suitable culture medium is MDl medium (peptone from casein,
tryptic (Merck), 0.3%; CaCl.sub.2.2H.sub.2 O, 0.05%; MgSO.sub.4.7H.sub.2
O, 0.2%; which is enriched with a carbohydrate source, e.g. glucose,
maltose, maltotriose, starch, cellulose, each 0.1%.
A further useful liquid culture medium contains peptone from casein, 0.1%;
CaCl.sub.2.2H.sub.2 O, 0.05%; MgSO.sub.4. 7H.sub.2 O, 0.2%; corn steep
powder or zein, 0.4%. The strain can also be cultivated in a defined
medium consisting of MgSO.sub.4.7H.sub.2 O, 0.15%; FeCl.sub.3. 6H.sub.2 O,
8 mg/l; KNO.sub.3, 0.2%; K.sub.2 HPO.sub.4, 0.025%; glucose.H.sub.2 O,
0.5%; CaCl.sub.2.2H.sub.2 O, 0.15%. Addition of 0.01-0.05% of peptone
results in homogeneous growth, an improved cell yield, and in increased
antibiotic production.
Culturing can be effected batchwise, e.g. by single or repeated addition of
nutrient solution, or continuously by continuous addition of nutrient
solution.
It is preferred to culture in several stages by preparing a preculture
(inoculum), e.g. in one of the cited culture media, with which preculture
the actual main culture is subsequently inoculated after fermentation for
one or two days, preferably in a dilution ratio of 1:10.
This preculture is obtained for example from a series of precultures. The
first culture of this series is obtained by growing the strain in question
for 14 days on a solid or liquid nutrient medium, e.g. agar+MD1+0.1% of
starch. A nutrient solution is then inoculated with the culture and
incubated for several days, e.g. 4-5 days. This nutrient solution can, if
desired, be used for inoculating a further nutrient solution, e.g.
MD1+0.1% of starch, or further nutrient solutions at intervals of 4-5
days, e.g. in a dilution ratio of about 3% (v/v), and the batch is
incubated under the stated conditions.
The course of the fermentation can be monitored analytically during the
fermentation, e.g. by measuring the pH value of the culture, which during
the fermentation falls from about 7.2 to about 6.0-6.5 and then rises to
about 7.5-8.0, or the optical density, which is a reference standard for
determining the growth of the particular strain, as well as
gravimetrically on the basis of the dry weight of the resultant biomass,
by thin-layer chromatography or by determining the antibiotic activity of
the components present in the culture filtrate.
The isolation of the compounds of formula I, especially of the main
component, sorangicin A, from the fermentation broth is effected by
methods known per se, having regard to the chemical, physical and
biological properties of the substances. Determination of the
concentration of antibiotics in the individual isolation steps, as well as
in the culture medium, can be made by thin-layer chromatography, e.g. on
silica gel (elution with e.g. methylene chloride/methanol), and/or by the
activity against different microorganisms, e.g. Staphylococcus aureus,
and/or by the inhibition of reverse transcriptase.
Two methods are suitable for isolating the compounds of formula I from the
crude fermentation broth:
a) Stirring the fermentation broth with macroporous non-ionic adsorber
resins, e.g. synthetic resins of aromatic structure, for example resins
based on polystyrene, e.g. styrene/divinylbenzene copolymers. Such resins
can be characterised by different customary statistical data such as pore
volume, specific surface area, average pore diameter, most frequent pore
diameter, pore size distribution, bead size distribution and the like.
Suitable adsorber resins have a pore volume of about 0.5 to 4.5 ml/g, a
specific surface area of about 100-1000 m.sup.2 /g, and an average pore
diameter of about 4 to 130 nm, and are commercially available under the
registered trademarks AMBERLITE XAD-1, XAD-2, XAD-4, XAD-1180 and ER-180
ex Rohm & Haas, DIAION HP-10, HP-20, HP-21, HP-30, EP-40, HP-50 ex
Mitsubishi, DUOLITE S-861, S-862, S-863 and ES 866 ex Dia-Prosim, IMAC Syn
46 and Syn 72 ex Akzo Chemie, KASTEL S-111, S-112, S-114 ex Montedison,
LEWATIT OC.1031 ex Bayer and RELITE ADS ex Resindion.
After separating the fermentation broth from the adsorber resin, e.g. by
sieving, the resin is washed with water and subsequently eluted with
increasing amounts of an organic solvent which is inert to the adsorber
resin, e.g. a mixture of water/methanol, with the bulk of the compounds of
formula I being eluted with 80-100% methanol. The eluates are concentrated
in vacuo and, as described for variant b), separated by HPLC.
b) The fermentation broth is separated from the biomass in conventional
manner, e.g. by filtration or centrifugation, and the filtrate is
extracted with a water-immiscible, or substantially water-immiscible,
solvent, e.g. methylene chloride, chloroform or, preferably, ethyl
acetate. The organic phase is evaporated under vacuum, affording a crude
extract. This crude extract is partitioned in a two-phase system
consisting of two immiscible organic solvents, e.g. methanol/heptane, and
the fermentation products, in particular the antibiotics of formula I,
become enriched in the polar phase. After removal of the polar solvent by
evaporation, the residue is dissolved in dilute ammonia solution and
washed with water-immiscible solvent, e.g. diethyl ether. The volatile
ammonia is removed under vacuum and the aqueous phase, after acidification
with an organic acid, e.g. formic acid or acetic acid, is extracted with
one of the water-immiscible, or substantially water-immiscible, solvents
mentioned above, e.g. methylene chloride. The resultant antibiotics are
subsequently purified by reversed phase chromatography, affording several
fractions which may contain sorangioside A and B in admixture or in pure
form, and sorangicin A and B in admixture or in pure form. The individual
components sorangioside A and B and sorangicin B are obtained by
separation of the respective fractions by HPLC, whereas the main
component, sorangicin A, can be obtained in crystalline form from the
appropriate fraction.
The main component, sorangicin A, can also be obtained by other variants of
these known separation methods, e.g. by using other solvents or mixtures
of solvents, or by using other chromatographic methods.
Salts of compounds of formula I can be prepared in a manner known per se.
Thus it is possible to form salts of compounds of formula I e.g. by
treatment with metal compounds such as alkali metal salts of suitable
organic carboxylic acids, e.g. the sodium salt of 2-ethylhexanoic acid, or
with an inorganic alkali metal salt or alkaline earth metal salt, e.g.
sodium bicarbonate, or with ammonia or a suitable organic amine, using the
salt-forming compound preferably in stoichiometric amount or only a small
excess thereof.
Salts can be converted in conventional manner into the free compounds,
metal and ammonium salts e.g. by treatment with a suitable acid.
Mixtures of stereoisomers, especially of diastereoisomers, can be separated
into the individual isomers in a manner known per se, e.g. by fractional
crystallisation, chromatography and the like.
Racemates can be resolved in a manner known per se, e.g. after converting
the optical antipodes into diastereoisomers, for example by reaction with
an optically active acid or base or with specific microorganisms.
The invention also relates to those embodiments of the process in which an
intermediate obtainable in any stage of the process is used as starting
material and the remaining steps are carried out therewith, or the process
is interrupted at any stage or a compound obtainable by the process of the
invention is prepared under the conditions of the process and further
processed in situ.
The pharmacologically acceptable compounds of the present invention can be
used e.g. for the preparation of pharmaceutical compositions which contain
an effective amount of a compound of formula I, preferably in admixture
with a significant amount of inorganic or organic, solid or liquid
pharmaceutically acceptable excipients. The invention also relates to such
pharmaceutical compositions and to the preparation and use thereof.
The pharmaceutical compositions of this invention are suitable for
parenteral, e.g. intravenous or intramuscular, administration, and, as
circumstances may require, also for oral administration or topical
application.
The compounds of formula I are used, for example, in the form of injectable
compositions, e.g. for intravenous administration, or of infusion
solutions. Such solutions are preferably isotonic aqueous solutions or
suspensions which can be prepared prior to use, e.g. from lyophilised
preparations which contain the active ingredient alone or together with a
carrier, e.g. mannitol. Pharmaceutical compositions for oral
administration may be sterilised and can contain adjuvants, e.g.
preservatives, stabilisers, wetting agents and/or emulsifiers,
solubilisers, salts for regulating the osmotic pressure, resorption
promoters and/or buffers. The pharmaceutical compositions of this
invention which, if desired, may contain further useful pharmacological
substances, e.g. other active ingredients, contain about 0.1 to 100%,
preferably about 1 to 100%, of active ingredient.
The pharmaceutical compositions are prepared in a manner known per se, e.g.
by conventional dissolving or lyophilising methods described in textbooks
of pharmacology.
UTILITY
Compounds of formula I, and the solvates or pharmaceutically acceptable
salts thereof, can be used as antibiotics in the form of pharmaceutical
compositions for the therapeutic treatment of the human or animal body,
for example as antibacterial antibiotics for the treatment of infections
caused by gram-positive or gram-negative bacteria. e.g. by Neisseria
gonorrhoeae or meningitidis, Staphylococcus aureus or epidermis,
streptococcae, including enterococcae, Haemophilus influenzae, Pseudomonas
aeruginosae, or anaerobic pathogens such as Bacteroides fragilis or
Clostridia spp., infections that are caused by bacteria that survive in
cells, e.g. leucocytes, for example staphylococcae or listeriae,
tuberculosis or infections that are caused by a typical mycobacteria such
as Mycobacterium intracellulare avium.
The compounds of this invention can also be used as antiviral antibiotics,
in particular for the treatment of infections caused by viruses with
reverse transcriptase activity, e.g. retroviruses (including HTLV III that
causes AIDS), or hepatitis viruses and the like. In addition to infections
with HTLV III (also named LAV I or HIV), other infections which can be
treated with the compounds of formula I are those with HTLV I, HTLV II and
HTLV IV (also named LAV II).
Depending on the nature, severity and duration of the infection, the
condition of the patient and the mode of administration, daily doses of
about 0.3 g to 10.0 g will be administered subcutaneously,
intramuscularly, intravenously, orally or by inhalation, for the treatment
of persons or warm-blooded animals having a body weight of 70 kg.
Further utilities exist in the field of plant protection, e.g. for
controlling bacterial diseases of greenhouse plants, and for the care of
infected trees and the like. Aerosols are suitable for such utilities.
The following Examples illustrate the experimental reduction to practice
and general operability of the present invention.
EXAMPLE 1
a) Preparation of the Inoculum of the Preculture
Bacteria of the strain So ce 12 in 100 ml of liquid MDl medium taken from
an approx. 14-day-old agar plate containing the medium MDl (0.3% of
peptone from casein, tryptic (ex Merck, Germany), 0.05% of
CaCl.sub.2.7H.sub.2 O, 0.2% of MgSO.sub.4.7H.sub.2 O) and 0.1% of starch
are inoculated with 0.1% of starch and the culture is incubated on a
gyratory table at 30.degree. C. and 160 rpm (revolutions per minute).
After incubation for 5 days, the culture is added to a fresh culture
medium MDl in a dilution ratio of 3% (v/v). Fresh nutrient medium MDl can
be inoculated as often as required in a dilution ratio of 3% (v/v), in
each case after incubating the preculture for 4 days. The last preculture
of this series is used in a dilution ratio of 10% (v/v) as inoculum for 4
l of a further preculture with MDl nutrient solution. This preculture is
incubated at 30.degree. C., stirred briefly and aerated under a weak flow
of air. After incubation for 2 days, a 70 l fermenter is inoculated with
this inoculum for the preparation of the preculture proper.
b) Preparation of the Preculture
70 l fermenter (available from Giovanola, Monthey VS, Switzerland),
equipped with paddle stirrer system. MDl medium (1.0 m) with 0.1% starch.
Temperature 30.degree. C. Stirring speed 320 rpm. Air supply: 0.3 Nm.sup.3
/h. The percentage of oxygen partial pressure falls in the course of the
fermentation and is 10-20% after 2 days. OD.sub.623 : c. 2. A 700 l
fermenter is inoculated with this preculture under sterile conditions.
c) Main Culture
700 l fermenter equipped with paddle stirrer system available from the same
firm as in b). Medium: 0.15% of magnesium sulfate heptahydrate, 8 mg/l of
iron(III) chloride hexahydrate, 0.05% of peptone, 0.2% of KNO.sub.3,
0.025% of K.sub.2 HPO.sub.4, 0.5% of glucose, 0.15% of calcium chloride
dihydrate; pH 7.3; initial stirring speed: 150 rpm, after 23 hours: 100
rpm. Percentage of oxygen partial pressure at the conclusion of the
fermentation: 10%. The fermentation is complete after 40 hours. Upon
conclusion of the fermentation, the supernatant has an inhibiting zone
diameter against Staphylococcus aureus of c. 14-15 mm. The biomass is
separated from the fermentation broth by centrifugation. The antibiotic
components are present in the culture filtrate. All process steps are
carried out under sterile conditions.
d) Working up of the Culture Filtrate
The culture filtrate is extracted with ethyl acetate. The solvent is then
evaporated, affording a crude extract. This extract is partitioned in a
two-phase system consisting of heptane/methanol, and the compounds of
formula I become enriched in the methanolic phase. The methanol is removed
by evaporation and the residue is dissolved in 12% aqueous ammonia
solution. The ammoniacal solution is washed repeatedly with ether and
freed from ammonia under vacuum. The aqueous phase is acidified with
formic acid and extracted with methylene chloride. The extract is
concentrated by evaporation and separation of the residue is effected by
reversed phase chromatography. Column: Labochrom PGC column 674.times.37
mm (available from Labomatic), packed with LiChroprep RP-18 (25-40 .mu.m),
ex Merck; eluant: methanol/buffer 68:33, buffer=0.5% formic acid in water,
adjusted to pH 7 with triethylamine. Rate of flow: 30 ml/min; detection:
UV absorption at 313 nm. The following fractions are obtained:
fraction 1: peak at t.sub.R =31 min: sorangiosid A+B
fraction 2: peak at t.sub.R =40 min: sorangiosid B
fraction 3: peak at t.sub.R =50 min: sorangicin A
fraction 4: peak at t.sub.R =69 min: sorangicin B
After removal of the organic solvent, the components present in the
individual fractions are extracted from the aqueous buffered phase with
dichloromethane or ethyl acetate.
Fraction 1: The mixture of soragioside A and B is separated by HPLC: column
250.times.16 mm (Knauer), packed with LiChrosorb Si 100 (10.mu.), ex
Merck; detection by UV absorption at 313 nm, eluant:
dichloromethane/heptane/isopropanol/conc. buffer (52:4:7:1), conc. buffer
consisting of a 1:2 mixture of methanol/formic acid neutralised with
triethylamine; rate of flow: 18 ml/min. Sorangioside B is obtained at a
retention time t.sub.R =9.4 min and sorangioside A at a retention time
t.sub.R =13.8 min.
Sorongioside A
HPLC: column 250.times.16 mm (Knauer), packed with Nucleosil 7 C.sub.6
H.sub.5 (ex Macherey-Nagel), eluant: methanol/water (65/35)+0.5% formic
acid; rate of flow: 10 ml/min; detection: UV absorption at 313 nm: t.sub.R
=26.4 min.
Thin-layer chromatography (silica gel Si 60 F 254 (Merck), eluant:
methylene chloride/methanol (8:2): R.sub.f =0.44.
[.alpha.].sub.D.sup.22 =+42.0 (c=0.9 in methanol).
UV (in methanol): .lambda..sub.max (1 g .epsilon.)=301 (4.34).
.sup.13 C-NMR, see Table.
Fraction 2: The fraction containing sorangioside B is purified by HPLC:
column 250.times.16 mm (Knauer), packed with Nucleosil 7 C.sub.6 H.sub.5
(ex Macherey-Nagel); detection: UV absorption at 131 nm; eluant:
methanol/water (70:30)+0.5% formic acid; rate of flow: 14 ml/min; t.sub.R
=10.2 min.
Sorongioside B
Thin-layer chromatography (silica gel Si 60 F 254 (Merck), eluant:
methylene chloride/methanol (8:2): R.sub.f =0.62.
[.alpha.].sub.D.sup.22 =+5.1 (c=1.8 in methanol).
UV (in methanol): .lambda..sub.max (1 g .epsilon.)=301 (4.41).
.sup.13 C-NMR, see Table.
Fraction 3: Crystallised from ethyl acetate.
Sorangicin A
m.p.: 105.degree.-107.degree. C.
[.alpha.].sub.D.sup.22 =+60.9 (c=0.7 in methanol).
UV (MeOH): .lambda..sub.max (1 g .epsilon.)=301 (4.33).
.sup.13 C-NMR see Table.
FAB-MS [neg. ions, xenon at 9 keV (Iontect), acceleration -8 kV,
post-acceleration 11 KV]: m/e=805 (M--H).sup.-, 897 (M--H+glycerol).sup.-.
EI-MS (70 eV, 245.degree.): m/e %=806 (M.sup.+, 0.3), 789 (0.6), 788 (1.5),
770 (0.8), 373 (2), 303 (2.5), 301 (3.5), 249 (44), 231 (5), 197 (15), 149
(20), 135 (21), 133 (22), 121 (35), 109 (40), 107 (43), 105 (100).
The compound is present in the form of the ethyl acetate. It can be
crystallized also from acetone.
Analysis: C.sub.47 H.sub.66 O.sub.11 .times.C.sub.4 H.sub.8 O theory C
68,43, H 8,33, O 23.23. found C 68,11, H 8,35, O 23.61.
Fraction 4: Purification by HPLC: column 250.times.16 mm (Knauer), packed
with Nucleosil 7 C.sub.6 H.sub.5 (ex Macherey-Nagel); detection: UV
absorption at 313 nm; eluant: methanol/water (75:25)+0.5% formic acid;
rate of flow: 14 ml/min; t.sub.R =11.7 min.
Sorangicin B
[.alpha.].sub.D.sup.22 =+49,1 (c=1.6 in methanol).
UV (methanol): .lambda..sub.max (1 g .epsilon.)=301 (4,41).
.sup.13 C-NMR see Table.
FAB-MS [neg. ions, xenon at 9 keV (Iontect), acceleration -8 kV,
post-acceleration 11 kV]: m/z=789 (M--H).sup.-, 881 (M--H+glycerol).sup.-.
EI-MS (70 eV, 270.degree.): m/z (%)=790 (M.sup.+,1), 773 (3), 772 (5), 755
(2), 754 (3), 643 (1), 250 (20), 249 (100), 197 (22), 191 (11), 181 (12),
169 (10), 163 (11), 161 (11), 159 (11), 149 (22), 133 (22), 123 (23), 121
(29), 107 (31), 105 (61).
High Resolution
C.sub.47 H.sub.66 O.sub.10 theory 790,4656 found 790,4618.
TABLE
______________________________________
.sup.13 C-NMR data of the sorangicins and sorangiosides in CD.sub.3 OD
No. Sorangioside.sup.d)
Sorangicin
C-Atom A B A B
______________________________________
1 178.01s 177.69s 177.85s
177.72s
2 35.38t 35.14t 35.28t
35.20t
3 26.27t 26.27t 26.29t
26.54t
4 28.20t 28.18t 28.20t
28.21t
5 38.52t 38.34t 38.51t
38.24t
6 32.90d 32.98d 32.96d
33.02d
7 a a a a
8 131.26s 131.68s 131.24s
131.48s
9 74.44d 74.72d 74.42d
74.45d
10 66.95d 67.03d 66.90d
66.91d
11 123.91d 123.70d 123.79d
123.68d
12 136.94d 136.89d 136.85d
136.86d
13 75.37d 74.72d 75.33d
75.07d
14 b b b b
15 a a a a
16 a a a a
17 b b b b
18 b b b b
19 136.41d 134.42d 134.36d
137.37d
20 127.23d 132.41d 130.16d
132.88d
21 83.21d 80.45d 74.36d
c
22 76.04d 43.05t 77.77d
45.08t
23 74.05d 71.10d 74.87d
c
24 31.40t 35.41t 30.86t
35.40t
25 70.98d 70.82d 71.07d
71.11d
26 38.03d 37.81d 38.51d
38.42d
27 74.44d 74.18d 75.05d
74.71d
28 b b b b
29 a a a a
30 a a a a
31 81.15d 81.06d 81.17d
81.09d
32 42.11d 42.02d 42.15d
42.10d
33 81.06d 80.94d 81.03d
80.95d
34 39.80t 39.93t 39.85t
39.92t
35 77.53d 77.12d 77.59d
77.35d
36 82.03d 81.77d 82.26d
82.13d
37 134.91d 134.77d 134.88d
134.66d
38 127.76d 127.22d 127.84d
127.55d
39 137.57d 137.70d 137.57d
137.72d
40 127.00d 126.82d 126.98d
126.89d
41 139.14d 139.39d 139.05d
139.21d
42 119.72d 119.44d 119.69d
119.49d
43 167.67s 167.33s 167.68s
167.51s
44 21.73q 21.54q 21.67q
21.54q
45 14.30q 14.24q 14.30q
14.23q
46 10.62q 10.62q 10.89q
10.82q
47 15.36q 15.32q 15.36q
15.33q
______________________________________
Footnotes:
a) sorangioside A: 134.28d, 133.77d, 133.06d, 132.41d, 128.31d;
sorangioside B: 135.06d, 133.76d, 133.11d, 132.41d, 128.40d;
sorangicine A: 134.15d, 133.61d, 133.00d, 132.78d, 128.34d;
sorangicine B: 134.27d, 133.53d, 133.03d, 132.70d, 128.40d;
b) sorangioside A: 37.06t, 35.38t, 34,37t, 33.61t;
sorangioside B: 37.01t, 35.41t, 33.85t, 33.64t;
sorangicine A: 37.12t, 35.45t, 33.97t, 33.35t;
sorangicine B: 37.13t, 35.40t, 33.49t, 33.49t;
c) 72.13d, 71.72d;
d) glucosyl signals: C1' C2' C3' C4' C5'
sorangioside A: 102.81d, 75.19d, 78.13d, 71.64d, 77.83d, 62.80t
sorangioside B: 102.62d, 75.27d, 78.21d, 71.61d, 77.73d, 62.83t
EXAMPLE 2
Dry-filled ampoules or vials containing 0.5 g of sorangiosin A as active
ingredient can be prepared as follows:
______________________________________
Composition: (for 1 ampoule or vial)
______________________________________
active ingredient
0.5 g
mannitol 0.05 g
______________________________________
A sterile aqueous solution consisting of active ingredient and mannitol is
filled under aseptic conditions into 5 ml ampoules or 5 ml vials, which
are then sealed and tested.
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